ABSTRACT
Laminar shear stress activates NADPH oxidase in vascular endothelial cells (ECs), and the generated superoxide radicals (O2(-.) are known to be involved in intercellular adhesion molecule (ICAM)-1 expression. In this study, the role of a glycosphingolipid (GSL), lactosylceramide (LacCer), as a second messenger in the shear-induced O2(-.) generation and ICAM-1 expression was examined. It is known that glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer) from ceramide, and subsequently lactosylceramide synthase (GalT-2) synthesizes LacCer from GlcCer. We observed that exposing cultured human umbilical vein ECs (HUVECs) to fluid shear stress (20 dyn/cm(2) for 30 min) activated GalT-2. Shear stress also increased EC O2(-.) generation, that peaked at 30 min, and surface ICAM-1 protein expression at 6 h post-shear. EC preincubation with the antioxidant N-acetylcysteine (NAC; 20 mM for 2 h) completely abolished the shear-induced O2(-.) production and significantly inhibited ICAM-1 expression. EC preincubation with D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of the GSL glycosyltransferases GlcT-1 and GalT-2, abrogated the shear-induced activation of GalT-2. D-PDMP also abolished the shear-induced O2(-.) production and ICAM-1 expression. We conclude that laminar shear stress activates GalT-2 to produce LacCer. In turn, LacCer activates NADPH oxidase, which produces O2(-.), and O2(-.) mediates the shear-induced increase in ICAM-1 expression. Thus, LacCer may play an important role in hemodynamic force-induced pathological conditions, such as atherosclerosis and ischemia/reperfusion injury.
Subject(s)
Antigens, CD , Endothelium, Vascular/metabolism , Intercellular Adhesion Molecule-1/metabolism , Lactosylceramides/pharmacology , Superoxides/metabolism , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Galactosyltransferases/metabolism , Humans , Morpholines/pharmacology , Stress, MechanicalABSTRACT
Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin and calcitonin (CT) are structurally and functionally related neuropeptides. It has recently been shown that the molecular pharmacology of CGRP and ADM is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Furthermore, RAMP proteins have also been shown to govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. In this study, we have cloned the rat RAMP family and characterized the pharmacology of rat CGRP and ADM receptors. Rat RAMP1, RAMP2 and RAMP3 shared 72%, 69% and 85% homology with their respective human homologues. As expected CRLR-RAMP1 coexpression conferred sensitivity to CGRP, whilst association of RAMP2 or RAMP3 with CRLR conferred high affinity ADM binding. Using specific oligonucleotides we have determined the expression of RAMP1, RAMP2 and RAMP3 mRNAs in the rat central nervous system by in situ hybridization. The localization of RAMP mRNAs was heterogeneous. RAMP1 mRNA was predominantly expressed in cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most abundant in hypothalamus; and RAMP3 was restrictively expressed in thalamic nuclei. Interestingly, in specific brain areas only a single RAMP mRNA was often detected, suggesting mutual exclusivity in expression. These data allow predictions to be made of where each RAMP protein may heterodimerize with its partner G-protein-coupled receptor(s) at the cellular level and consequently advance current understanding of cellular sites of action of CGRP, ADM, amylin and CT. Furthermore, these localization data suggest that the RAMP family may associate and modify the behaviour of other, as yet unidentified neurotransmitter receptors.
Subject(s)
Central Nervous System/metabolism , Membrane Proteins/genetics , Neuropeptides/metabolism , RNA, Messenger/metabolism , Receptors, Calcitonin/metabolism , Adrenomedullin , Amino Acid Sequence/physiology , Amyloid/metabolism , Animals , Calcitonin/metabolism , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Receptor-Like Protein , Cells, Cultured , Cloning, Molecular , DNA, Complementary/isolation & purification , Diencephalon/metabolism , Intracellular Signaling Peptides and Proteins , Islet Amyloid Polypeptide , Male , Membrane Proteins/metabolism , Mesencephalon/metabolism , Molecular Sequence Data , Peptides/metabolism , Rats , Rats, Sprague-Dawley , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Protein 2 , Receptor Activity-Modifying Protein 3 , Receptor Activity-Modifying Proteins , Rhombencephalon/metabolism , Sequence Homology, Amino Acid , Spinal Cord/metabolism , Telencephalon/metabolismABSTRACT
The precise involvement of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) receptors in the pleiotropic actions of 5-HT remain incompletely known. To gain insights into their physiological function(s), localization of mRNAs encoding these subtypes was carried out using in situ hybridization on rat brain sections. Localization was heterogeneous. For example, 5-ht(5A) mRNA was widely expressed while 5-ht(5B) mRNA was predominantly expressed in habenula, hippocampus and inferior olive. 5-ht(6) mRNA was abundant in olfactory tubercles and caudate putamen, and highest levels of 5-HT(7) mRNA were observed in multiple thalamic nuclei. These data suggest that these receptors may have distinct functional roles within the serotonergic system.
Subject(s)
Brain Chemistry/physiology , Receptors, Serotonin/genetics , Animals , Gene Expression/physiology , Habenula/chemistry , Habenula/physiology , Hippocampus/chemistry , Hippocampus/physiology , In Situ Hybridization , Male , Neostriatum/chemistry , Neostriatum/physiology , Olfactory Pathways/chemistry , Olfactory Pathways/physiology , Olivary Nucleus/chemistry , Olivary Nucleus/physiology , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, Serotonin/analysisABSTRACT
5-Hydroxytryptamine exerts modulatory physiological effects on both the central and peripheral nervous systems by activation of discrete receptor families. 5-ht(5A) is among the recently cloned, novel 5-HT receptors and currently under investigation to identify its pharmacological characteristics and potential physiological function(s). In this study, antibodies raised to a 5-ht(5A)-specific peptide were characterized using dot blot, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry, and the distribution of 5-ht(5A)-like immunoreactive material determined in rat brain. A major band of 41 kDa was observed following SDS-PAGE, corresponding to the predicted size of this receptor. 5-ht(5A)-like immunoreactivity was detected in areas known to have significant serotonergic input, such as hypothalamic and amygdaloid nuclei. Interestingly, 5-ht(5A)-like immunoreactivity showed a predilection for the suprachiasmatic nucleus, suggesting its possible role in the regulation of circadian rhythms, a function previously ascribed to 5-HT(1A) and 5-HT(7) receptors.
Subject(s)
Brain Chemistry , Receptors, Serotonin/analysis , Animals , Antibody Specificity , Diencephalon/chemistry , Male , Rats , Rats, Sprague-Dawley , Receptors, Serotonin/immunology , Rhombencephalon/chemistry , Telencephalon/chemistryABSTRACT
Recent cloning studies have isolated proteins which confer responsiveness to calcitonin gene-related peptide (CGRP). In this study, we have determined the central nervous system (CNS) distribution of the mRNA of one such protein, termed CGRP-receptor component protein (RCP), by in situ hybridization. CGRP-RCP mRNA was widely expressed in the guinea-pig CNS, being particularly abundant in cerebellum and hippocampus. These data should assist in the determination of the potential physiological function(s) of this protein in the CNS.
